This invention relates generally to the field of constructs and methods for producing viral vectors, and more particularly, for the production of adenoviruses.
Recombinant adenoviruses have been described as useful for delivery of transgenes to cells for a variety of purposes, including both therapeutic and prophylactic (vaccine) uses. However, successful commercialization of E1-deleted adenoviruses will require suitable manufacturing processes, which have yet to be developed. Infection of an E1 trans-complementing cell line with the vector and purification of the resulting lysate is a simple and scalable process that yields sufficient quantities of product. Unfortunately, production of E1-deleted adenovirus vectors for gene therapy has been plagued by emergence of replication competent adenovirus (RCA) caused by homologous recombination between the vector and transfected E1 gene.
Several strategies have been described to avoid RCA. However, to date none of these approaches has resulted in an E1-complementing cell line which is stable and produces high yields of E1-defective adenoviruses in the absence of detectable RCA.
J. -L. Imler et al., Gene Ther., 3:75-84 (1996) describes an A549 cell stably transfected with E1a and E1b open reading frames (ORFs) and contiguous pIX gene. The E1a was driven by phosphoglycerate kinase promoter and RCA was reportedly eliminated. However, more recent publications describing this system reveal that Imler was unable to detect E1b protein expression. See, Introgene, WO 97/00326, published Jan. 3, 1997.
This Introgene application described an alternative system to that of Imler, cited above. This application describes cell lines derived from certain human diploid cells with E1a and E1b expressed, but no pIX (ECACC NO. 96022940). The cells were produced by transfection of human embryonic retinoblast (HER) cells with a vector containing nt 459-3510 of Ad, which corresponds to E1a, E1b, but excludes the E1a promoter, a portion of the E1b gene encoding the E1b 8.3 kb protein, and any pIX sequences.
Another system for avoiding RCA is described in Massie, U.S. Pat No. 5,891,690. The patent describes an Ad E1-complementing cell line having a stably integrated complementation element comprising a portion of the Ad E1 region covering the E1a gene and the E1b gene, but lacking the 5xe2x80x2 ITR, the packaging sequence, and the E1a promoter. Further, the E1a gene is under control of a first promoter element and the E2b gene is under control of a second promoter. A specific cell line described and claimed by Massie contains nt 532-3525 of Ad5, which includes E1a, the E1b promoter, and a portion of the E1b gene. This cell line does not contain the carboxy terminus of the E1b gene, which encodes the 8.3 kb product, nor does it contain pIX gene sequences.
What is needed in the art is a stable E1-complementing cell line, which expresses all adenoviral E1a and E1b gene products, and which produces high yields of E1-defective adenoviruses in the absence of detectable RCA.
Advantageously, the present invention provides E1 expressing cell lines that are stable, can be adapted to suspension culture in serum free medium, and yields high quantity of vector. Significantly, the cell lines allow isolation and subculture of E1-deleted recombinant adenoviruses in an environment free of replication competent adenovirus (RCA). Further, the cell lines of the invention effectively plaque vector to allow isolation and subculture of new recombinants in an environment free of RCA.
Thus, in one aspect, the invention provides an E1-complementing cell line useful for production of recombinant E1-defective adenoviruses in the absence of detectable replication-competent adenovirus. The E1-complementing cell line contains an aneuploid cell line stably transformed with a nucleic acid molecule comprising nucleic acid sequences encoding adenovirus E1a and adenovirus E1b under the control of a phosphoglycerate kinase (PGK) promoter. Suitably, the nucleic acid molecule lacks adenovirus sequences 5xe2x80x2 to the sequences encoding adenovirus E1a.
In another aspect, the invention provides a method for packaging of E1-defective adenoviral particles in the absence of replication competent adenovirus. The method involves introducing a vector into cells from the E1-complementing cell line of the invention, where the vector contains a defect in the adenovirus E1 region, adenovirus 5xe2x80x2 and 3xe2x80x2 cis-elements necessary for replication and packaging, adenovirus pIX, and regulatory sequences necessary for expression of the adenoviral genes and transgene.
In another aspect, the invention provides a method of producing E1-defective adenoviral particles in the absence of detectable replication competent adenovirus. The method involves infecting cells from the E1-complementing cell line of the invention with an E1-defective adenovirus and culturing under conditions which permit the cell to express the E1a and E1b proteins.
Other aspects and advantages of the invention will be readily apparent from the following detailed description of the invention.